Microparticles and nanoparticles having negative surface charges

ABSTRACT

This invention provides methods for producing a polymer particle which contains unusually high negative charges on the surface of the particle. Preferably, the polymer is pharmaceutically acceptable. The negative charges can be conferred by chemical groups such as carboxyl, sulfonate, nitrate, fluorate, chloride, iodide, persulfate, and many others, with carboxyl group being preferred. The invention also provides polymer particle produced by the methods of the invention.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/916,439, filed Mar. 3, 2016, which is a US National stage entry ofInternational Application No. PCT/US2013/073019, which designated theUnited States and was filed on Dec. 4, 2013, published in English, whichclaims the benefit of U.S. Provisional Application No. 61/733,216, filedon Dec. 4, 2012. The entire teachings of the above applications areincorporated herein by reference.

BACKGROUND OF THE INVENTION

Certain carboxylated particles, such as carboxylated polystyrene, PLGA,or diamond particles, when administered to subjects, may amelioratecertain conditions, such as pathological inflammatory immune responses(see WO 2012/065153).

Inflammatory diseases and disorders are conditions in which an abnormalor otherwise deregulated inflammatory response contributes to theetiology or severity of disease. Examples include autoimmune diseasessuch as rheumatoid arthritis, multiple sclerosis, and diabetes,infectious diseases such as tuberculosis and various forms of meningitisand encephalitis including West Nile Virus encephalitis and otherdisorders include atherosclerosis and ischemic reperfusion.

Many of these diseases are characterized by a mononuclear cellinfiltration at a site of tissue injury or other insult. Examples ofmononuclear cells that have been observed in these infiltrations includelymphocytes, especially T lymphocytes, and cells of the mononuclearphagocyte system (MPS cells) such as monocytes, macrophages, dendriticcells, microglial cells and others.

However, it is Applicant's belief that carboxylated PLGA particlesproduced using conventional means are frequently not biocompatible andthus PLGA particles resulting from such manufacturing processes may notbe safe for use on humans and animals. In addition, it is Applicant'sbelief that PLGA particles produced using conventional means may notcontain sufficient number of COOH groups for attaching API's or otherchemical entities to microparticles and nanoparticles.

There is a need to prepare negatively charged (e.g., carboxylated PLGA)microparticles and nanoparticles with enhanced therapeutic properties.

SUMMARY OF THE INVENTION

One aspect of the invention provides a method for the preparation of acomposition comprising microparticles or nanoparticles having negativesurface charges, said method comprising producing said microparticles ornanoparticles with a pharmaceutically acceptable polymer using either anemulsion process or a precipitation process: (a) wherein said emulsionprocess or said precipitation process is carried out in an aqueoussolution having a pH that promotes ionization of said pharmaceuticallyacceptable polymer; (b) wherein said pharmaceutically acceptable polymerhas an average molecular weight of from about 500 to about 1,000,000 Da,about 500 to about 50,000 Da, or preferably from about 1,000 to about50,000 Da, or about 1,000 to about 30,000 Da; and/or, (c) wherein saidpharmaceutically acceptable polymer contains multiple negatively chargedterminal groups.

In certain embodiments, the emulsion process comprises: (1) dissolvingthe pharmaceutically acceptable polymer in a first solvent to form apolymer solution; (2) emulsifying the polymer solution in a solution ofa second solvent to form an emulsion, wherein the first solvent is notmiscible or partially miscible with the second solvent, and wherein thesolution of the second solvent optionally comprises a pharmaceuticallyacceptable negatively charged agent; and, (3) removing the first solventto form said microparticles or nanoparticles having negative surfacecharges; wherein said solution of the second solvent is optionally saidaqueous solution.

In certain embodiments, the precipitation process comprises: (1)dissolving the pharmaceutically acceptable polymer in a first solvent toform a polymer solution; (2) preparing a solution of a second solvent,wherein the first solvent is miscible with the second solvent, andwherein the solution of the second solvent optionally comprises apharmaceutically acceptable negatively charged agent and optionallycomprises a surfactant; and, (3) combining (e.g., adding) the polymersolution to the solution of the second solvent while mixing, thusforming said microparticles or nanoparticles having negative surfacecharges; wherein said solution of the second solvent is optionally saidaqueous solution.

In certain embodiments, the emulsion process is a double emulsionprocess comprising: (1) dissolving the pharmaceutically acceptablepolymer in a first solvent to form a polymer solution; (2) adding asmall amount of a solution of a second solvent to the polymer solutionto form a mixture, wherein the first solvent is not miscible orpartially miscible with the second solvent, and wherein the solution ofthe second solvent optionally comprises an active pharmaceuticalingredient (API); (3) emulsifying the mixture to form a first emulsion;(4) emulsifying the first emulsion in the solution of the second solventto form a second emulsion, wherein the solution of the second solventoptionally comprises a pharmaceutically acceptable negatively chargedagent, and optionally further comprises a surfactant; and, (5) removingthe first solvent to form said microparticles or nanoparticles havingnegative surface charges; wherein said solution of the second solvent isoptionally said aqueous solution.

In certain embodiments, the pharmaceutically acceptable negativelycharged agent is incorporated into said microparticles or nanoparticlesto increase negative surface charges on said microparticles ornanoparticles.

In certain embodiments, the pharmaceutically acceptable negativelycharged agent is incorporated into said microparticles or nanoparticlesto increase the numbers of carboxyl groups on said microparticles ornanoparticles.

In certain embodiments, the pharmaceutically acceptable negativelycharged agent comprises polyacrylic acid, or poly(ethylene-alt-maleicacid) (PEMA).

In certain embodiments, the pharmaceutically acceptable polymer is anaturally occurring polymer.

In certain embodiments, the naturally occurring polymer is cellulose,dextrin, hyluronic acid, gelatin, polysaccharide, amino acid, orpolyhydroxyalkanoates.

In certain embodiments, the pharmaceutically acceptable polymer is asynthetic polymer.

In certain embodiments, the synthetic polymer is polyacrylic acid,polymethacrylic acid, polylactic acid, polyglycolic acid,polyhydroxybutytic acid, polylactide, polyglycolide,poly(lactide-co-glycolide) polycaprolactone, polyanhydride, orpoly(lactide-co-glycolide) or PLGA, or a salt, derivative, copolymer, ormixture thereof. Preferably, the synthetic polymer is a biodegradablepolymer.

In certain embodiments, the synthetic polymer is a PLGA polymer havingan L/G ratio of from about 95/5 to 5/95, preferably from 85/15 to 15/85,and most preferably about 50/50.

In certain embodiments, the microparticles or nanoparticles have a zetapotential of from about −5 mV to about −200 mV, preferably from about−15 mV to about −100 mV, most preferably from −35 mV to −85 mV.

In certain embodiments, the first solvent is a volatile solvent.

In certain embodiments, the polymer is a PLGA polymer, and the volatilesolvent is methylene chloride, ethyl acetate, or chloroform.

In certain embodiments, the solution of the second solvent comprises asurfactant.

In certain embodiments, said solution of the second solvent is a mixtureof the first and second solvent. In certain embodiments, the volumeratio of the first solvent to the second solvent in said solution of thesecond solvent is about 0.1:99.9, about 1:99, about 5:95, about7.5:92.5; about 7.8:92.2; about 8:92, or about 10:90.

In certain embodiments, the surfactant comprises organic or inorganicpharmaceutical excipients; various polymers; oligomers; naturalproducts; nonionic, cationic, zwitterionic, or ionic surfactants; andmixtures thereof.

In certain embodiments, the polymer is a PLGA polymer, and thesurfactant is/comprises polyvinyl alcohol, polyvinylpyrrolidone, a Tweenseries surfactant, Pluronic F-68, Poloxamer series, or Triton X-100 andits derivatives.

In certain embodiments, the emulsifying step comprises homogenization,mechanical stirring, and/or microfluidization.

In certain embodiments, the first solvent is removed through solventexchange and/or evaporation.

In certain embodiments, the pharmaceutically acceptable negativelycharged agent is a carboxyl-containing agent.

In certain embodiments, the carboxyl-containing agent compriseshyaluronic acid; gelatin; polysaccharide; polyacrylic acid;polymethacrylic acid; hydroxyethylmethacrylic acid; amino acid; or asalt, derivative, copolymer, or mixture thereof.

In certain embodiments, the pH that promotes ionization of thepharmaceutically acceptable polymer is between about 4-14, 6-14, 6-10,or about 8-12.

In certain embodiments, a base (e.g., sodium hydroxide, potassiumhydroxide, sodium bicarbonate, sodium carbonate, potassium bicarbonate,or potassium carbonate) is used to adjust pH of the aqueous solution.

In certain embodiments, the second solvent is water, and the firstsolvent miscible with water is or comprises acetone, tetrahydrofuron(THF), acetonitrile, dimethyl sulfoxide (DMSO), or dimethylformamide(DMF).

In certain embodiments, the multiple negatively charged terminal groupsare carboxyl terminal groups.

In certain embodiments, the polymer containing multiple negativelycharged carboxyl terminal groups is produced by using acarboxyl-functional initiator in the preparation of the polymer.

In certain embodiments, the carboxyl-functional initiator is anα-hydroxyl acid, preferably lactic acid or glycolic acid.

In certain embodiments, the polymer containing multiple negativelycharged carboxyl terminal groups is produced by grafting acarboxyl-containing entity to the polymer.

In certain embodiments, the carboxyl-containing entity is or comprisespolyacrylic acid, polymethacrylic acid, poly(hydroxyethyl methacrylicacid), poly(maleic acid), polyanhydrides, or a salt, derivative,copolymer, or mixture thereof.

In certain embodiments, the polymer containing multiple negativelycharged carboxyl terminal groups is produced by converting a functionalgroup on the polymer to carboxyl group.

In certain embodiments, the functional group is a hydroxyl group, andwherein the hydroxyl group is converted to the carboxyl group byreacting with an anhydride (e.g., dihydrofuran-2,5-dione).

In certain embodiments, the polymer containing multiple negativelycharged carboxyl terminal groups is produced by using an initiator thatcontains multiple carboxyl groups for generating hyperbranched polymercontaining multiple negatively charged carboxyl terminal groups.

In certain embodiments, the microparticles or nanoparticles have averageparticle sizes of from about 1 nm to about 1000 μm, preferably fromabout 10 nm to about 100 μm, more preferably from about 20 nm to about 5μm, and most preferably from about 50 nm to about 2 μm.

In certain embodiments, the pharmaceutically acceptable polymer is PLGA,and wherein the microparticles or nanoparticles are PEGylated.

In certain embodiments, the microparticles or nanoparticles arePEGylated by mixing polyethylene glycol (PEG) or PEG-containing entityduring the preparation of the microparticles and nanoparticles.

In certain embodiments, the microparticles or nanoparticles arePEGylated by using copolymers of PEG and PLGA.

In certain embodiments, the microparticles or nanoparticles arePEGylated by physically absorbing PEG polymers or polymers containingPEG units onto the PLGA microparticles and nanoparticles.

In certain embodiments, the microparticles or nanoparticles arePEGylated by conjugating PEG units to the surface of the PLGAmicroparticles or nanoparticles via covalent bonds.

In certain embodiments, an API (active pharmaceutical ingredient) iscovalently attached to the surface of the microparticles ornanoparticles via covalent bonds.

In certain embodiments, the method further comprises chemicallyconjugating a biomolecule (e.g., a peptide or a protein) to the surfaceof the microparticles or nanoparticles.

Another aspect of the invention provides a composition comprisingmicroparticles or nanoparticles having negative surface charges, whereinthe composition is prepared according to any one of the methodsdescribed herein.

In certain embodiments, the composition is free from other API (e.g.,attached peptide or antigenic moieties).

Another aspect of the invention provides a pharmaceutical compositioncomprising any of the subject composition, and a pharmaceuticallyaccepted carrier or excipient.

Another aspect of the invention provides a method of treating a diseaseor condition in a subject, wherein the disease or condition is treatablewith microparticles or nanoparticles with negative surface charge,comprising administering a composition or a pharmaceutical compositioncomprising the microparticles or nanoparticles to the subject, therebytreating the disease or condition.

In certain embodiments, the disease or condition is characterized by aninflammatory immune response.

In certain embodiments, the disease or condition is multiple sclerosis(MS), psoriasis, rheumatoid arthritis, type 1 diabetes.

In certain embodiments, the method further comprises administering asecond therapeutic agent known to be effective for treating the diseaseor condition.

It should be understood that any embodiments described herein can becombined with any other embodiments, including embodiments describedonly under one aspect of the invention, and embodiments described onlyin the examples.

DETAILED DESCRIPTION OF THE INVENTION

This invention described herein provides polymer particles(microparticles and nanoparticles) which contain unusually high or atleast increased negative charges on the surface of said particlescompared to those produced using conventional methods. Preferably,polymers used for the preparation of the particles are pharmaceuticallyacceptable materials.

As used herein, “pharmaceutically acceptable” includes those compounds,materials, compositions, and/or dosage forms which are, within the scopeof sound medical judgment, suitable for medical or veterinary use whenin contact with the tissues of human beings and animals, without causingexcessive toxicity, irritation, allergic response, or other problems orcomplications, commensurate with a reasonable benefit/risk ratio.Preferably, a pharmaceutically acceptable material (e.g., polymer ormicroparticles/nanoparticles produced therefrom) is suitable or approvedfor human medical use.

As used herein, “microparticles” are roughly round, sphere, orsphere-like in shape, and are generally within the size range of, e.g.,between about 1-1,000 μm, or between about 10-100 μm. The subjectmicroparticles may also include particles that are less likely to clumpin vivo.

As used herein, “nanoparticles” are roughly round, sphere, orsphere-like in shape, and are generally within the size range of, e.g.,between about 1-1,000 nm, between about 10-1,000 nm, or between about50-1,000 nm, or between about 100-500 nm. The subject nanoparticles mayalso include particles that are less likely to clump in vivo.

It is not necessary that each microparticle or nanoparticle be uniformin size, although they are generally of a size sufficient to triggerphagocytosis in an antigen presenting cell (APC) or other MPS cell. Thusin one embodiment, the subject microparticles and nanoparticles have adiameter sufficient to trigger phagocytosis in an antigen presentingcell (APC) or other MPS cell.

As used herein, “about” generally means up to ±10% of the particularterm being modified.

The negative charge can be, for example, in the form of a carboxylate,sulfonate, nitrate, fluorate, chloride, iodide, persulfate, and manyother negatively charged chemical groups. In certain embodiments, thenegative charge is mainly conferred by carboxyl groups. The subjectmicroparticles or nanoparticles having net negative surface charges, andmay or may not contain some positive surface charges.

A preferred pharmaceutically acceptable polymer useful for thepreparation of the subject microparticles and nanoparticles is PLGA.PLGA is typically prepared by ring-opening polymerization of lactide andglycolide. In this reaction, Stannous octoate is usually used as thecatalyst, although other catalysts may also be used. An initiator, suchas an alcohol, is often used to initiate the polymerization reaction. Ifno initiator is intentionally added, trace amount of polar compoundcontaining an active proton, such as alcohol and water, may serve as theinitiator. Polymerization usually results in a PLGA polymer with acarboxyl group at the chain terminal, as illustrated below:

R—OH+L(lactide monomer)+G(glycolide monomer)=PLGA-COOH

Therefore, each PLGA polymer molecule is typically linear, and typicallycontains a single COOH group at the chain terminal. Consequently,conventional PLGA particles prepared from such PLGA polymers only havesmall amount of COOH groups on the surface, and the negative chargethereon may not be sufficient for certain uses, such as treatinginflammatory diseases. In addition, there may not be sufficient numbersof COOH groups for covalently attaching API's or other chemical moietysuch as protein ligands or other targeting agents to the surface of saidmicroparticles and nanoparticles. Such protein ligands or othertargeting agents may bind to a receptor or a binding partner on thesurface of a target cell, tissue, organ, or location.

The instant invention provides various methods or combinations thereoffor producing PLGA particles with additional negatively charged groups(e.g., carboxyl groups) on the PLGA particle surfaces. Such PLGAparticles with increased net negative surface charges are particularlyuseful, for example, to treat certain diseases (such as inflammatorydiseases) and to facilitate the conjugation of API's or other chemicalentity to the microparticles and nanoparticles.

The invention described herein provides several basic methods for thepreparation of particles with highly negative surface charges. Thesemethods are not mutually exclusive, and may be combined with one anotherto produce additive or even synergistic effects to producemicroparticles and nanoparticles with highly negatively chargedsurfaces.

Thus in one aspect, the invention provides a method for the preparationof a composition comprising microparticles or nanoparticles havingnegative surface charges, the method comprising producing themicroparticles or nanoparticles with a pharmaceutically acceptablepolymer using either an emulsion process or a precipitation process,wherein the method comprises any one or more features described below,or combination thereof.

Specifically, one feature of the methods of the invention comprisescarrying out the emulsion process or the precipitation process in anaqueous solution having a pH that promotes ionization of thepharmaceutically acceptable polymer. For example, the pharmaceuticallyacceptable polymer may comprise a carboxyl group that becomes ionized(e.g., carries a negative charge) at a basic pH. In another example, thepharmaceutically acceptable polymer may comprise a chemical moietyhaving a low pKa such that the moiety becomes ionized at a relativelyacidic pH (e.g., pH 5 or 6).

While not wishing to be bound by any particular theory, the ionizedgroups or moieties, compared to their non-ionized forms, tend more to beexposed on the surface of the eventually formed microparticles ornanoparticles, and tend less to be buried inside the eventually formedmicroparticles or nanoparticles.

Another feature of the methods of the invention comprises usingpharmaceutically acceptable polymers having a low average molecularweight. As described herein, PLGA is typically prepared by ring-openingpolymerization of lactide and glycolide using Stannous octoate as thecatalyst and an alcohol as an initiator. Polymerization usually resultsin a linear PLGA polymer with a single carboxyl group at the chainterminal. Thus, by using a PLGA polymer having lower molecular weights,or shorter polymer chains, relatively higher carboxyl group density canbe reached in the nanoparticles and microparticles. Here, carboxyl groupdensity can be defined as number of carboxyl groups per gram of polymer.

In certain embodiments, the average molecular weight of thepharmaceutically acceptable polymer is within a desired range.

The low end of the range is preferably no less than about 100, 200, 300,400, 500, 600, 700, 800, 900, 1000, 1200, 1500, 2000, 2500, or 3000 Da.The desired range can have a low end of any of the above values.

The high end of the range is preferably no more than 50,000, 40,000,35,000, 30,000, 25,000, 20,000, 15,000, 10,000, 7,500, or 5,000 Da. Thedesired range can have a high end of any of the above values.

For instance, the desired range may be from about 500 to about 50,000Da, or from about 1,000 to about 30,000 Da.

In certain pharmaceutically acceptable polymers, such as PLGA, averagemolecular weight is expressed in other physical properties such asinherent viscosity. Inherent Viscosity (IV) is a viscometric method formeasuring molecular size. IV is based on the flow time of a polymersolution through a narrow capillary relative to the flow time of thepure solvent through the capillary. For certainly measures in theinstant application, the solvent used is typically chloroform, and thepolymer concentration is about 0.5% (w/v). The temperature at which theviscosity is measured is about 30° C. The units of IV are typicallyreported in deciliters per gram (dL/g). Thus, for example, the desiredpharmaceutically acceptable polymer (such as PLGA) that may be used inthe instant invention may have an inherent viscosity of from about 0.01to about 20 dL/g, or from about 0.05 to about 2.0 dL/g.

Yet another feature of the methods of the invention comprises usingpharmaceutically acceptable polymers containing multiple (i.e., two ormore, ≥2, ≥3, ≥4, ≥5, ≥10, ≥20, ≥50, ≥75, ≥100, or a range between anyof the two recited values, etc.) potentially negatively charged terminalgroups. In certain embodiments, the multiple negatively charged terminalgroups are carboxyl terminal groups.

Polymers containing multiple carboxyl groups can be obtained by avariety of means, including: 1) using a carboxyl-functional initiator inthe preparation of the polymer. Common carboxyl-functional initiatorsinclude but are not limited to α-hydroxyl acid. For example, lacticacid, glycolic acid; 2) grafting carboxyl-containing entities to thepolymer chain; 3) converting other functional groups on the PLGA polymerto carboxyl groups via a chemical reaction. For example, hydroxyl groupson PLGA polymers may be converted to carboxyl groups by reacting thehydroxyl groups with an anhydride (e.g., dihydrofuran-2,5-dione). Anexemplary reaction is depicted in the scheme below:

-   -   and, 4) using hyperbranched PLGA polymers that contain multiple        carboxyl-containing arms obtained by, for example, using an        initiator that contains multiple carboxyl groups on its        molecule.

The number of carboxyl group on each (PLGA) polymer is preferably from 1to 100, more preferably from 2 to 10 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or10).

The methods of the invention can comprise any one or more featuresdescribed herein.

Any art-recognized emulsion process may be used in the methods of theinvention. In certain embodiments, the subject microparticles andnanoparticles (e.g., PLGA microparticles and nanoparticles) can beprepared by an emulsification process comprising the following steps(not necessarily in this order): 1) dissolving the pharmaceuticallyacceptable polymer (e.g., PLGA) in a first solvent (e.g., methylenechloride) to form a polymer solution; 2) emulsifying the polymersolution (e.g., PLGA solution) in a solution of a second solvent (e.g.,an aqueous solution, or an organic solvent) to form an emulsion, whereinthe first solvent is not miscible or partially miscible with the secondsolvent, and wherein the solution of the second solvent optionallycomprises a pharmaceutically acceptable negatively charged agent; and,3) removing the first solvent to form the microparticles ornanoparticles having negative surface charges, wherein the solution ofthe second solvent is optionally the aqueous solution.

In certain embodiments, in the emulsification process, the weight ratioof the PLGA solution to the aqueous solution is typically from 1:1,000to 10:1, preferable from 1:100 to 1:1.

As used herein, miscibility is defined to be the property of liquids tomix in all proportions, forming a homogeneous solution.Substances/liquids are said to be immiscible or not miscible, if in someproportion, they do not form a solution.

Exemplary solvents miscible with water include acetone, tetrahydrofuron(THF), acetonitrile, dimethyl sulfoxide (DMSO), dimethylformamide (DMF).

Another art-recognized emulsion process is commonly known as doubleemulsion process, which may be particularly useful when an activepharmaceutical ingredient (API), such as a protein-based therapeuticsprepared in an aqueous solution, is first emulsified with apharmaceutically acceptable polymer solution to form a first emulsionsuch that the protein-based therapeutics is encapsulated within thepolymer solution. Then the polymer, and the therapeutics encapsulatedtherein, is again emulsified in a larger volume of solvent to form asecond emulsion (e.g., the water-in-oil-in-water or w/o/w type doubleemulsion), before the microparticle or nanoparticle is formed.

For example, in the above described w/o/w technique, a relatively smallamount of aqueous protein solution may be introduced into a relativelylarger amount of organic solvent, such as methylene chloride or ethylacetate, that dissolves the hydrophobic polymer PLGA. The first emulsionis then formed using a suitable method, e.g., probe sonication orhomogenization. After formation of the first emulsion, a second emulsionis formed by introducing the first emulsion into an aqueous solutioncontaining an emulsifier, e.g., polyvinyl alcohol. Again, ahomogenization method is used to form the second emulsion. This is nextfollowed by a period of solvent evaporation leading to the hardening ofthe polymer, typically by stirring for some hours. As a result, theprotein solution is trapped into the relative hydrophobic matrix of thePLGA polymer forming small inclusions. Finally, the microparticlesornanoparticles formed are collected, washed (e.g., with distilledwater) via repeated centrifugation or filtration, followed bydehydration, typically by lyophilization.

Thus in certain embodiments, the subject microparticles andnanoparticles (e.g., PLGA microparticles and nanoparticles) can beprepared by a double emulsification process comprising the followingsteps (not necessarily in this order): 1) dissolving thepharmaceutically acceptable polymer (e.g., PLGA) in a first solvent(e.g., methylene chloride) to form a polymer solution; 2) adding arelatively small amount of a solution of a second solvent into thepolymer solution to form a mixture, wherein the first solvent is notmiscible or partially miscible with the second solvent, and wherein thesolution of the second solvent optionally comprises an activepharmaceutical ingredient (API); 3) emulsifying the mixture to form afirst emulsion; 4) emulsifying the first emulsion in the solution of thesecond solvent to form a second emulsion, wherein the solution of thesecond solvent optionally comprises a pharmaceutically acceptablenegatively charged agent, and optionally further comprises a surfactant;and, 5) removing the first solvent to form said microparticles ornanoparticles having negative surface charges; wherein said solution ofthe second solvent is optionally said aqueous solution.

In certain embodiments, the volume of the small amount of the solutionof the second solvent added to the polymer solution for the generationof the first emulsion is typically from 0.01% to 50%, preferable from0.1% to 10%, based on the volume of the PLGA solution.

In certain embodiments, the volume ratio of the first emulsion to thesolution of the second solvent described as in Step 4) above istypically from 10:1 to 1:10,000, preferably from 1:1 to 1:100.

Any art-recognized precipitation process may be used in the methods ofthe invention. In certain embodiments, the subject microparticles andnanoparticles (e.g., PLGA microparticles and nanoparticles) can beprepared by a precipitation process comprising the following steps (notnecessarily in this order): 1) dissolving the pharmaceuticallyacceptable polymer (e.g., PLGA) in a first solvent (e.g., acetone) toform a polymer solution; 2) preparing a solution of a second solvent(e.g., aqueous solution, such as 1 mM NaOH solution), wherein the firstsolvent is miscible with the second solvent, and wherein the solution ofthe second solvent optionally comprises a pharmaceutically acceptablenegatively charged agent and optionally comprises a surfactant; and, 3)adding the polymer solution to the solution of the second solvent whilemixing, thus forming the microparticles or nanoparticles having negativesurface charges; wherein the solution of the second solvent isoptionally the aqueous solution.

In certain embodiments, in the precipitation process, the volume ratioof the PLGA solution to the aqueous solution is typically from 10:1 to1:1,000, preferably from 1:1 to 1:10.

In certain embodiments, as an alternative procedure to Step 3) in theprecipitation process, the solution of the second solvent (e.g., theaqueous solution) can be added to the polymer solution (e.g., PLGAsolution).

In any of the above embodiments, the pharmaceutically acceptablenegatively charged agent may cover the surface of the microparticles ornanoparticles, and/or be at least partially incorporated into saidmicroparticles or nanoparticles to increase negative surface charges onthe microparticles or nanoparticles. Representative pharmaceuticallyacceptable negatively charged agent may comprise polyacrylic acid, orpoly(ethylene-alt-maleic acid) (PEMA).

In any of the aspects described above, while numerous types ofcarboxylated particles can ameliorate inflammatory immune responses, itis necessary to use particles that are made of materials that arepharmaceutically acceptable. These pharmaceutically acceptable polymersmay be of natural or synthetic origin. Examples of naturally occurringpolymers include but are not limited to cellulose, dextrin, hyaluronicacid, gelatin, polysaccharides, amino acid, polyhydroxyalkanoates, etc.

Synthetic polymers useful for the instant invention are typicallybiocompatible and/or biodegradable, and are therefore safe for human andanimal use. Examples of the synthetic polymers useful for the inventioninclude polymers such as polyacrylic acid, polymethacrylic acid,polylactic acid, polyglycolic acid, polyhydroxybutytic acid,polylactide, polyglycolide, poly(lactide-co-glycolide) polycaprolactone,polyanhydrides, etc. Poly(lactide-co-glycolide) or PLGA is a preferredmaterial for the preparation of the subject negatively chargedparticles.

The composition and biodegradability of the subject PLGA polymer ispartly determined by the molar ratio of lactide (L) to glycolide (G)unit in the polymer, or L/G ratio. The L/G ratio of the PLGA polymer inthe present invention can be from 100/0 to 0/100. As used herein, an L/Gratio of “100/0” refers to polylactide or PLA, and an L/G ratio of“0/100” refers to polyglycolide, or PGA. Preferably the L/G ratio forthe PLGA polymer is from about 95/5 to 5/95, more preferably from about85/15 to 15/85. The most preferable L/G ratio in the present inventionis about 50/50.

Other polymers can be mixed with the PLGA polymer in the preparation ofthe PLGA microparticles and nanoparticles. For example, polyethyleneglycol, or PEG, is often added to the PLGA for enhanced performance.PEGylated particles are useful because they often have increasedcirculation time in human or animal bodies.

In certain embodiments, copolymers of PEG and PLGA can also be used.

The microparticles and nanoparticles prepared from the PEG and PLGAmixture or PEG and PLGA copolymer are referred to as PEGylated PLGAmicroparticles and nanoparticles.

Such “PEGylation” process can also be done after microparticles andnanoparticles are formed. In this case, PEG polymers or other polymerscontaining PEG units are coated via physical absorption onto the PLGAmicroparticles and nanoparticles.

The PEG units can also be attached to the surface of PLGA microparticlesor nanoparticles via covalent bonds. Such process is often referred toas “conjugation.” In a conjugation process, a reactive entity containingPEG units react with certain functional groups on the surface of themicroparticles and nanoparticles to form chemical bonds.

Thus in certain embodiments, the pharmaceutically acceptable polymer isPLGA, and the microparticles or nanoparticles are PEGylated. Themicroparticles or nanoparticles may be PEGylated by mixing polyethyleneglycol (PEG) or PEG-containing entity during the preparation of themicroparticles and nanoparticles. The microparticles or nanoparticlesmay also be PEGylated by using copolymers of PEG and PLGA. Themicroparticles or nanoparticles can further be PEGylated by physicallyabsorbing PEG polymers or polymers containing PEG units onto the PLGAmicroparticles and nanoparticles. The microparticles or nanoparticlesmay additionally be PEGylated by conjugating PEG units to the surface ofthe PLGA microparticles or nanoparticles via covalent bonds.

The pharmaceutically acceptable negatively charged agent may be apharmaceutically acceptable carboxyl-containing agent, such as oneuseful for producing (PLGA) microparticles and nanoparticles withadditional amount of carboxyl groups on the surface. Suchcarboxyl-containing agent includes but is not limited to hyaluronic acidor analogs or derivative thereof, gelatin, polysaccharides,hydroxyethylmethacrylic acid, polyacrylic acid, polymethacrylic acid,amino acids, or their salts, derivatives, copolymers and mixtures.

The amount of the pharmaceutically acceptable negatively charged agentused in the current invention is from 0.01% to 30%, preferably from 0.1%to 5%, based on the weight of the pharmaceutically acceptable polymer(such as PLGA) used in the formulation.

Hyaluronic acid analogs include many natural polysaccharides that havebeen sulphated, which may behave like sulphated glycosaminoglycan, suchas heparin (Hoffman et al., 1982, Carbohydrate Res., 2:115; Kindness etal., 1980, Brit. J. Pharmac., 69:675; Horton et al., 1973, CarbohydrateRes., 30:349; Okada et al., 1979, Makromol. Chem. 180:813; Kikuchi etal., 1979, Nippon Kagaku Kaishi, 1:127; Manzac et al., 1981, Proc. ThirdM.I.S.A.O., 5:504). Moreover, sulphuric, carboxy or sulphonated groupshave been attached to synthetic polymers such as polystyrene (Kanmaugueet al., 1985, Biomaterials, 6:297) and polyurethanes (Ito et al., 1992,Biomaterials, 13:131), which may be used as HA analogs in the instantinvention. The high density of the negative charges on these HA analogs(e.g., the N-sulphated group of the glucosamine residues), which may bepH-dependent, confers additional benefit when added to the subjectmicro-/nano-particles as HA analogs.

The HA analogs can be produced by chemical reactions known for thesulphation of polysaccharides (see, e.g., WO 88/00211; EP 0340628;Carbohydrate Research, 158:183-190, 1986).

An important family of HA analogs include HA derivatives produced bymodification of hyaluronic acid.

Certain hyaluronic acid derivatives are known in the art. For example,WO 95/25751 (incorporated by reference) describes various heparin-likesulphated polysaccharide derivatives, such as sulphated hyaluronic acidof different molecular weight ranges, such that the number of sulfategroups per repetitive unit is in the range of from 0.5 to 3.5. Thesulphated HA not only contains more negative charges per repeat unit,but also inhibits the production of tumor necrosis factor (TNF) when thesulphated hyaluronic acid having a molecular weight in the range ofbetween about 10,000 and about 50,000 Daltons is used. Excessive TNFαactivity is associated with the proliferation of inflammatory cells, andis the cause of many inflammatory disease conditions. Thus using suchsulfated hyaluronic acid can further enhance the anti-inflammatoryeffect of the subject micro-/nano-particles.

Preferably, the sulfated hyaluronic acid has a molecular weight in therange between about 10,000 and about 50,000 Daltons, or between about50,000 and about 250,000 Daltons, or between about 250,009 and about750,000 Daltons, or between about 750,000 and about 1, 250,000 Daltons,wherein in each case, the degree of sulfation of said sulfatedhyaluronic acid is 2.5, 3.0 or 3.5 sulfate groups per repetitive unit ofhyaluronic acid.

WO 1998/045335A1 describes certain biocompatible sulphated compounds ofhyaluronic acid and derivatives thereof, optionally salified, whereinthe glucosamines are partially N-sulphated, or partially N-sulphated andpartially or totally O-sulphated in position 6. Specifically, such HAderivatives are obtained by means of a controlled sulphation reaction ofthe amino group of the glucosamine of hyaluronic acid, previouslyN-deacetylated according to the procedure described by P. Shaklee (1984)Biochem. 1, 217:187-197 (incorporated herein). Apart from theirbiocompatibility characteristics, such N-sulphated derivatives also hasantiviral activity, anti-inflammatory activity, antithrombotic andanticoagulant properties.

In certain embodiments, the degree of sulphation per one dimeric unit ofthe amino groups varies between 1 and 70% and that of the hydroxyl groupin position 6 varies between 0 and 100%. In certain embodiments, thedegree of sulphation per one dimeric unit of the amino groups variesbetween 5 and 40% and that of the hydroxyl group in position 6 variesbetween 0 and 100%.

Additional hyaluronic acid derivatives are described in U.S. Pat. No.7,993,678, which derivatives have at least one hydroxyl-group ofhyaluronic acid is substituted, through a reaction with aryl/alkylsuccinic anhydrides (ASA), to produce aryl/alkyl succinic anhydride HAderivatives. The derivative carries more negative charge per repeatunit, and can be used with or in place of HA in the methods of theinvention.

In certain embodiments, the hyaluronic acid analogs or derivatives areno more than 10, 15, 20, 25, or 30% (w/w) of the pharmaceuticallyacceptable polymer.

In certain embodiments, the microparticles or nanoparticles have anegative (surface) charge. The negative charge density on thecarboxylated microparticles and nanoparticles can be quantified by zetapotential. The zeta potential of the carboxylated microparticles andnanoparticles is typically measured in an aqueous suspension of theparticles at a pH of from 4 to 10, preferably from 5 to 8. In certainembodiments, the microparticles or nanoparticles produced by the methodsof the invention may have a zeta potential of from about −5 mV to about−200 mV, preferably from about −15 mV to about −100 mV, most preferablyfrom −35 mV to −85 mV.

The solvent used in the dissolving step for the polymer can be any typeof solvent that dissolves the polymer (e.g., PLGA). However, a volatilesolvent is preferably used for its removal. For example, preferredsolvents for forming the PLGA solution include methylene chloride, ethylacetate, and chloroform.

In the emulsifying step, the (aqueous) solution may contain a surfactantor surface stabilizer. Surfactants generally include compounds thatlower the surface tension of a liquid, the interfacial tension betweentwo liquids, or that between a liquid and a solid. Surfactants may actas detergents, wetting agents, emulsifiers, foaming agents, anddispersants. Surfactants are usually organic compounds that areamphiphilic, which contain both hydrophobic groups (usually branched,linear, or aromatic hydrocarbon chain(s), fluorocarbon chain(s), orsiloxane chain(s) as “tail(s)”) and hydrophilic groups (usually heads).Surfactants are most commonly classified according to their polar headgroup: a non-ionic surfactant has no charge groups in its head; an ionicsurfactant carries a net charge—if the charge is negative, thesurfactant is anionic, and if the charge is positive, it is cationic. Ifa surfactant contains a head with two oppositely charged groups, it istermed zwitterionic. In certain embodiments, anionic or zwitterionicsurfactants, such as those containing carboxyl groups (“carboxylates”),are preferably used in the instant invention. The carboxylates are themost common surfactants and comprise the alkyl carboxylates, such assodium stearate, sodium lauroyl sarcosinate, and carboxylate-basedfluorosurfactants such as perfluorononanoate, perfluorooctanoate (PFOAor PFO).

While not wishing to be bound by any particular theory, surfactant maybe useful for the formation and stabilization of the emulsion droplets.The surfactant may also comprise organic or inorganic pharmaceuticalexcipients, various polymers, oligomers, natural products, nonionic,cationic, zwitterionic, or ionic surfactants, and mixtures thereof.

The surfactants that can be used for the preparation of the subject(PLGA) microparticles/nanoparticles include polyvinyl alcohol,polyvinylpyrrolidone, Tween series, Pluronic series, Poloxamer series,Triton X-100, etc. Additional suitable surfactants are provided hereinbelow.

The emulsification process may be carried out by any art-recognizedmeans, such as homogenization, mechanical stirring, ormicrofluidization, etc.

The removal of solvent is usually achieved by, for example, solventexchange and evaporation.

In certain embodiments, in order to ensure that most carboxyl groups arepresent on the surface of the subject (e.g., PLGA) microparticles andnanoparticles, the aqueous solution is adjusted to a pH that promotesionization of a moiety on the polymer, such as a basic pH for a carboxylgroup on PLGA. The pH is preferably in the range of about 4-14, 6-14,6-10, or about 8-12, depending on the pKa of the polymer group that canbecome ionized to carry a negative charge. The pH of the aqueoussolution can be adjusted to the preferred range by adding, for example,a base or a solution thereof, such as sodium hydroxide, potassiumhydroxide, sodium bicarbonate, sodium carbonate, potassium bicarbonate,potassium carbonate, etc.

The size of the subject microparticles and nanoparticles is from about 1nm to about 1000 μm, preferably from about 10 nm to about 100 μm, andmost preferably from about 20 nm to about 5 μm. For example, themicroparticles and nanoparticles may have an average size of about 100,300, 500, or 700 nm.

As used herein, particle size can be determined by any conventionalparticle size measuring techniques well known to those skilled in theart. Such techniques include, for example, sedimentation field flowfractionation, photon correlation spectroscopy, light scattering,dynamic light scattering, light diffraction, and disk centrifugation.

Combinations of more than one surfactant can be used in the invention.Useful surfactants or surface stabilizers which can be employed in theinvention may include, but are not limited to, known organic andinorganic pharmaceutical excipients. Such excipients include variouspolymers, low molecular weight oligomers, natural products, andsurfactants. Surfactants or surface stabilizers include nonionic,cationic, zwitterionic, and ionic surfactants.

Representative examples of other useful surfactants or surfacestabilizers include hydroxypropyl methylcellulose,hydroxypropylcellulose, polyvinylpyrrolidone, sodium lauryl sulfate,sodium dioctylsulfosuccinate, gelatin, casein, lecithin (phosphatides),dextran, gum acacia, cholesterol, tragacanth, stearic acid, benzalkoniumchloride, calcium stearate, glycerol monostearate, cetostearyl alcohol,cetomacrogol emulsifying wax, sorbitan esters, polyoxyethylene alkylethers (e.g., macrogol ethers such as cetomacrogol 1000),polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fattyacid esters (e.g., the commercially available Tweens® such as e.g.,Tween 20® and Tween 80® (ICI Specialty Chemicals)); polyethylene glycols(e.g., Carbowaxs 3550® and 934® (Union Carbide)), polyoxyethylenestearates, colloidal silicon dioxide, phosphates, carboxymethylcellulosecalcium, carboxymethylcellulose sodium, methylcellulose,hydroxyethylcellulose, hydroxypropylmethylcellulose phthalate,noncrystalline cellulose, magnesium aluminum silicate, triethanolamine,polyvinyl alcohol (PVA), 4-(1,1,3,3-tetramethylbutyl)-phenol polymerwith ethylene oxide and formaldehyde (also known as tyloxapol,superione, and triton), poloxamers (e.g., Pluronics F68® and F108®,which are block copolymers of ethylene oxide and propylene oxide);poloxamines (e.g., Tetronic 908®, also known as Poloxamine 908®, whichis a tetrafunctional block copolymer derived from sequential addition ofpropylene oxide and ethylene oxide to ethylenediamine (BASF WyandotteCorporation, Parsippany, N.J.)); Tetronic 1508® (T-1508) (BASF WyandotteCorporation), Tritons X-200®, which is an alkyl aryl polyether sulfonate(Rohm and Haas); Crodestas F-110®, which is a mixture of sucrosestearate and sucrose distearate (Croda Inc.);p-isononylphenoxypoly-(glycidol), also known as Olin-10G® or Surfactant10-G® (Olin Chemicals, Stamford, Conn.); Crodestas SL-40(Croda, Inc.);and SA9OHCO, which is C18H37CH2(CON(CH3)-CH2(CHOH)4(CH2OH)2 (EastmanKodak Co.); decanoyl-N-methylglucamide; n-decyl β-D-glucopyranoside;n-decyl β-D-maltopyranoside; n-dodecyl β-D-glucopyranoside; n-dodecylβ-D-maltoside; heptanoyl-N-methylglucamide;n-heptyl-p-D-glucopyranoside; n-heptyl β-D-thioglucoside; n-hexylβ-D-glucopyranoside; nonanoyl-N-methylglucamide; n-noylβ-D-glucopyranoside; octanoyl-N-methylglucamide;n-octyl-β-D-glucopyranoside; octyl β-D-thioglucopyranoside;PEG-derivatized phospholipid, PEG-derivatized cholesterol,PEG-derivatized cholesterol derivative, PEG-derivatized vitamin A,PEG-derivatized vitamin E, lysozyme, random copolymers of vinylpyrrolidone and vinyl acetate, and the like.

Examples of useful cationic surfactants or surface stabilizers include,but are not limited to, polymers, biopolymers, polysaccharides,cellulosics, alginates, phospholipids, and nonpolymeric compounds, suchas zwitterionic stabilizers, poly-n-methylpyridinium, anthryulpyridinium chloride, cationic phospholipids, chitosan, polylysine,polyvinylimidazole, polybrene, polymethylmethacrylatetrimethylammoniumbromide bromide (PMMTMABr), hexyldesyltrimethylammoniumbromide (HDMAB), polyvinylpyrrolidone-2-dimethylaminoethyl methacrylatedimethyl sulfate, 1,2Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine-N4Amino(PolyethyleneGlycol)2000] (sodium salt) (also known as DPPE-PEG(2000)-Amine Na)(Avanti Polar Lipids, Alabaster, Al), Poly(2-methacryloxyethyltrimethylammonium bromide) (Polysciences, Inc., Warrington, Pa.) (alsoknown as S1001), poloxamines such as Tetronic 908®, also known asPoloxamine 908®, which is a tetrafunctional block copolymer derived fromsequential addition of propylene oxide and ethylene oxide toethylenediamine (BASF Wyandotte Corporation, Parsippany, N.J.),lysozyme, long-chain polymers such as alginic acid, carrageenan (FMCCorp.), and POLYOX (Dow, Midland, Mich.).

Other useful cationic stabilizers include, but are not limited to,cationic lipids, sulfonium, phosphonium, and quaternary ammoniumcompounds, such as stearyltrimethylammonium chloride,benzyl-di(2-chloroethyl)ethylammonium bromide, coconut trimethylammonium chloride or bromide, coconut methyl dihydroxyethyl ammoniumchloride or bromide, decyl triethyl ammonium chloride, decyl dimethylhydroxyethyl ammonium chloride or bromide, C12-15dimethyl hydroxyethylammonium chloride or bromide, coconut dimethyl hydroxyethyl ammoniumchloride or bromide, myristyl trimethyl ammonium methyl sulphate, lauryldimethyl benzyl ammonium chloride or bromide, lauryl dimethyl(ethenoxy)4 ammonium chloride or bromide, N-alkyl (C12-18)dimethylbenzylammonium chloride, N-alkyl (C14-18)dimethyl-benzyl ammonium chloride,N-tetradecylidmethylbenzyl ammonium chloride monohydrate, dimethyldidecyl ammonium chloride, N-alkyl and (C12-14) dimethyl 1-napthylmethylammonium chloride, trimethylammonium halide, alkyl-trimethylammoniumsalts and dialkyl-dimethylammonium salts, lauryl trimethyl ammoniumchloride, ethoxylated alkyamidoalkyldialkylammonium salt and/or anethoxylated trialkyl ammonium salt, dialkylbenzene dialkylammoniumchloride, N-didecyldimethyl ammonium chloride,N-tetradecyldimethylbenzyl ammonium, chloride monohydrate,N-alkyl(C12-14) dimethyl 1-naphthylmethyl ammonium chloride anddodecyldimethylbenzyl ammonium chloride, dialkyl benzenealkyl ammoniumchloride, lauryl trimethyl ammonium chloride, alkylbenzyl methylammonium chloride, alkyl benzyl dimethyl ammonium bromide, C12, C15, C17trimethyl ammonium bromides, dodecylbenzyl triethyl ammonium chloride,poly-diallyldimethylammonium chloride (DADMAC), dimethyl ammoniumchlorides, alkyldimethylammonium halogenides, tricetyl methyl ammoniumchloride, decyltrimethylammonium bromide, dodecyltriethylammoniumbromide, tetradecyltrimethylammonium bromide, methyl trioctylammoniumchloride (ALIQUAT 336TM), POLYQUAT 10TM, tetrabutylammonium bromide,benzyl trimethylammonium bromide, choline esters (such as choline estersof fatty acids), benzalkonium chloride, stearalkonium chloride compounds(such as stearyltrimonium chloride and Di-stearyldimonium chloride),cetyl pyridinium bromide or chloride, halide salts of quaternizedpolyoxyethylalkylamines, MIRAPOLTM and ALKAQUATTM (Alkaril ChemicalCompany), alkyl pyridinium salts; amines, such as alkylamines,dialkylamines, alkanolamines, polyethylenepolyamines,N,N-dialkylaminoalkyl acrylates, and vinyl pyridine, amine salts, suchas lauryl amine acetate, stearyl amine acetate, alkylpyridinium salt,and alkylimidazolium salt, and amine oxides; imide azolinium salts;protonated quaternary acrylamides; methylated quaternary polymers, suchas poly[diallyl dimethylammonium chloride] and poly-[N-methyl vinylpyridinium chloride]; and cationic guar.

Such exemplary cationic surfactants or surface stabilizers and otheruseful cationic surfactants or surface stabilizers are described in J.Cross and E. Singer, Cationic Surfactants: Analytical and BiologicalEvaluation (Marcel Dekker, 1994); P. and D. Rubingh (Editor), CationicSurfactants: Physical Chemistry (Marcel Dekker, 1991); and J. Richmond,Cationic Surfactants: Organic Chemistry, (Marcel Dekker, 1990), each ofwhich is incorporated by reference herein in its entirety.

Nonpolymeric cationic surfactants or surface stabilizers are anynonpolymeric compound, such as benzalkonium chloride, a carboniumcompound, a phosphonium compound, an oxonium compound, a haloniumcompound, a cationic organometallic compound, a quaternary phosphorouscompound, a pyridinium compound, an anilinium compound, an ammoniumcompound, a hydroxylammonium compound, a primary ammonium compound, asecondary ammonium compound, a tertiary ammonium compound, andquaternary ammonium compounds of the formula NR1R2R3R4(+). For compoundsof the formula NR1R2R3R4(+): (i) none of R1-R4 are CH3; (ii) one ofR1-R4 is CH₃; (iii) three of R1-R4 are CH3; (iv) all of R1-R4 are CH3;(v) two of R1-R4 are CH3, one of R1-R4 is C6H5CH2, and one of R1-R4 isan alkyl chain of seven carbon atoms or less; (vi) two of R1-R4 are CH3,one of R1-R4 is C6H5CH2, and one of R1-R4 is an alkyl chain of nineteencarbon atoms or more; (vii) two of R1-R4 are CH3 and one of R1-R4 is thegroup C6H5 (CH2)n, where n>1; (viii) two of R1-R4 are CH3, one of R1-R4is C6H5CH2, and one of R1-R4 comprises at least one heteroatom; (ix) twoof R1-R4 are CH3, one of R1-R4 is C6H5CH2, and one of R1-R4 comprises atleast one halogen; (x) two of R1-R4 are CH3, one of R1-R4 is C6H5CH2,and one of R1-R4 comprises at least one cyclic fragment; (xi) two ofR1-R4 are CH3 and one of R1-R4 is a phenyl ring; or (xii) two of R1-R4are CH3 and two of R1-R4 are purely aliphatic fragments.

Such compounds include, but are not limited to, behenalkonium chloride,benzethonium chloride, cetylpyridinium chloride, behentrimoniumchloride, lauralkonium chloride, cetalkonium chloride, cetrimoniumbromide, cetrimonium chloride, cethylamine hydrofluoride,chlorallylmethenamine chloride (Quaternium-15), distearyldimoniumchloride (Quaternium-5), dodecyl dimethyl ethylbenzyl ammoniumchloride(Quaternium-14), Quaternium-22, Quaternium-26, Quaternium-18hectorite, dimethylaminoethylchloride hydrochloride, cysteinehydrochloride, diethanolammonium POE (10) oletyl ether phosphate,diethanolammonium POE (3)oleyl ether phosphate, tallow alkoniumchloride, dimethyl dioctadecylammoniumbentonite, stearalkonium chloride,domiphen bromide, denatonium benzoate, myristalkonium chloride,laurtrimonium chloride, ethylenediamine dihydrochloride, guanidinehydrochloride, pyridoxine HCl, iofetamine hydrochloride, megluminehydrochloride, methylbenzethonium chloride, myrtrimonium bromide,oleyltrimonium chloride, polyquaternium-1, procainehydrochloride,cocobetaine, stearalkonium bentonite, stearalkoniumhectonite, stearyltrihydroxyethyl propylenediamine dihydrofluoride, tallowtrimoniumchloride, and hexadecyltrimethyl ammonium bromide.

Most of these surfactants or surface stabilizers are knownpharmaceutical excipients and are described in detail in the Handbook ofPharmaceutical Excipients, published jointly by the AmericanPharmaceutical Association and The Pharmaceutical Society of GreatBritain (The Pharmaceutical Press, 2000), specifically incorporated byreference.

The surfactants or surface stabilizers are commercially available and/orcan be prepared by techniques known in the art.

In certain embodiments, the surface of the subject microparticle ornanoparticle is composed of a material that minimizes nonspecific orunwanted biological interactions between the particle surface and theinterstitium, e.g., the particle surface may be coated with a materialto prevent or decrease non-specific interactions. Steric stabilizationby coating particles with hydrophilic layers such as poly(ethyleneglycol) (PEG) and its copolymers such as PLURONICS (including copolymersof poly(ethylene glycol)-bl-poly(propylene glycol)-bl-poly(ethyleneglycol)) may reduce the non-specific interactions with proteins of theinterstitium as demonstrated by improved lymphatic uptake followingsubcutaneous injections.

In yet another embodiment, particles of the present invention may alsocontain additional components. For example, carriers may have imagingagents incorporated or conjugated to the carrier. An example of acarrier nanosphere having an imaging agent that is currentlycommercially available is the Kodak X-sight nanospheres. Inorganicquantum-confined luminescent nanocrystals, known as quantum dots (QDs),have emerged as ideal donors in FRET applications: their high quantumyield and tunable size-dependent Stokes Shifts permit different sizes toemit from blue to infrared when excited at a single ultravioletwavelength. (Bruchez et al., Science, 1998, 281:2013; Niemeyer, C. M.,Angew. Chem. Int. Ed., 2003, 42:5796; Waggoner, A. Methods Enzymol.,1995, 246:362; Brus, L. E., J. Chem. Phys., 1993, 79, 5566). Quantumdots, such as hybrid organic/inorganic quantum dots based on a class ofpolymers known as dendrimers, may be used in biological labeling,imaging, and optical biosensing systems (Lemon et al., J. Am. Chem.Soc., 2000, 122:12886). Unlike the traditional synthesis of inorganicquantum dots, the synthesis of these hybrid quantum dot nanoparticlesdoes not require high temperatures or highly toxic, unstable reagents.(Etienne et al, Appl. Phys. Lett., 87:181913, 2005).

Another aspect of the invention provides a composition comprising thesubject microparticles or nanoparticles having negative surface charges,wherein the composition is prepared according to any one of the subjectmethods described herein or combinations thereof.

In certain embodiments, the composition is free from other activepharmaceutical ingredients or API, such as attached peptide or antigenicmoieties.

In certain other embodiments, the composition comprises an API, and theAPI is covalently attached to the surface of the microparticles ornanoparticles via covalent bonds, such as a bond formed between an amidegroup of a protein and a carboxyl group on the surface of themicroparticle or nanoparticle.

In certain embodiments, the amount of the API may be about 0.01-50%(w/w) of the microparticle or nanoparticle, or about 0.05-25%, about0.1-10%, about 0.2-5%, 0.5-3%, 1-5%, or 2-5% (w/w) of the microparticleor nanoparticle.

In certain other embodiments, the composition comprises, in place of anAPI, a targeting moiety, such as a peptide or protein ligand or domain,covalently attached to the surface of the microparticles ornanoparticles, which targeting moiety specifically or preferentiallybinds to a target site (such as a cell surface receptor or bindingpartner for the targeting moiety), such that the micro- or nanoparticlebearing such a targeting moiety will be specifically or preferentiallydirected to the target site in vivo. The targeting moiety bearing micro-or nanoparticle may further comprise an API that is encapsulated orembedded within the micro- or nanoparticle that can be released orotherwise effective at the target site.

A related aspect of the invention provides a pharmaceutical compositioncomprising the subject composition, and a pharmaceutically acceptedcarrier or excipient. Pharmaceutical compositions are described below inmore details in a separate section.

A further aspect of the invention provides a method of treating adisease or condition in a subject in need thereof, or a method ofreducing the duration or severity of the disease or condition in thesubject in need thereof, wherein the disease or condition is treatablewith microparticles or nanoparticles with negative surface charge,comprising administering a composition or a pharmaceutical compositioncomprising the microparticles or nanoparticles to the subject, therebytreating the disease or condition.

In a related aspect, the invention provides a method of regulating animmune response in a subject in need thereof, preferably a mammal, morepreferably a human, comprising administering a composition or apharmaceutical composition comprising the microparticles ornanoparticles to the subject, thereby regulating the immune response.Methods of immunoregulation provided by the invention include those thatsuppress and/or inhibit an innate immune response or an adaptive immuneresponse, including, but not limited to, an immune response stimulatedby immunostimulatory polypeptides or viral or bacterial components. Thesubject particles are administered in an amount sufficient to regulatethe immune response. As described herein, regulation of an immuneresponse may be humoral and/or cellular, and is measured using standardtechniques in the art and as described herein.

In certain embodiments, the disease or condition is characterized by aninflammatory immune response.

Treatable diseases or conditions include, but are not limited to: anautoimmune disorder, such as multiple sclerosis, scleroderma, type-Idiabetes, rheumatoid arthritis, thyroiditis, systemic lupuserythmatosis, Reynauud's syndrome, Sjorgen's syndrome, autoimmuneuveitis, autoimmune myocarditis, or Crohn's disease. In a particularembodiment, the autoimmune disease is multiple sclerosis. An individualhaving an autoimmune disease or inflammatory disease is an individualwith a recognizable symptom of an existing autoimmune disease orinflammatory disease.

Autoimmune diseases can be divided in two broad categories:organ-specific and systemic. Autoimmune diseases include, withoutlimitation, rheumatoid arthritis (RA), systemic lupus erythematosus(SLE), type I diabetes mellitus, type II diabetes mellitus, multiplesclerosis (MS), immune-mediated infertility such as premature ovarianfailure, scleroderma, Sjogren's disease, vitiligo, alopecia (baldness),polyglandular failure, Grave's disease, hypothyroidism, polymyositis,pemphigus vulgaris, pemphigus foliaceus, inflammatory bowel diseaseincluding Crohn's disease and ulcerative colitis, autoimmune hepatitisincluding that associated with hepatitis B virus (HBV) and hepatitis Cvirus (HCV), hypopituitarism, graft-versus-host disease (GvHD),myocarditis, Addison's disease, autoimmune skin diseases, uveitis,pernicious anemia, and hypoparathyroidism.

Autoimmune diseases may also include, without limitation, Hashimoto'sthyroiditis, Type I and Type II autoimmune polyglandular syndromes,paraneoplastic pemphigus, bullus pemphigoid, dermatitis herpetiformis,linear IgA disease, epidermolysis bullosa acquisita, erythema nodosa,pemphigoid gestationis, cicatricial pemphigoid, mixed essentialcryoglobulinemia, chronic bullous disease of childhood, hemolyticanemia, thrombocytopenic purpura, Goodpasture's syndrome, autoimmuneneutropenia, myasthenia gravis, Eaton-Lambert myasthenic syndrome,stiff-man syndrome, acute disseminated encephalomyelitis, Guillain-Barrésyndrome, chronic inflammatory demyelinating polyradiculoneuropathy,multifocal motor neuropathy with conduction block, chronic neuropathywith monoclonal gammopathy, opsoclonus-myoclonus syndrome, cerebellardegeneration, encephalomyelitis, retinopathy, primary biliary sclerosis,sclerosing cholangitis, gluten-sensitive enteropathy, ankylosingspondylitis, reactive arthritides, polymyositis/dermatomyositis, mixedconnective tissue disease, Behcet's syndrome, psoriasis, polyarteritisnodosa, allergic anguitis and granulomatosis (Churg-Strauss disease),polyangiitis overlap syndrome, hypersensitivity vasculitis, Wegener'sgranulomatosis, temporal arteritis, Takayasu's arteritis, Kawasaki'sdisease, isolated vasculitis of the central nervous system,thromboangiutis obliterans, sarcoidosis, glomerulonephritis, andcryopathies. These conditions are well known in the medical arts and aredescribed, for example, in Harrison's Principles of Internal Medicine,14th edition, Fauci, A. S. et al., Eds., New York: McGraw-Hill, 1998.

In another embodiment, the diseases or conditions include an allergicdisorder or condition, such as allergic disease, allergy, eczema,asthma, allergic rhinitis or skin hypersensitivity. An individual havingan allergic disease or asthma is an individual with a recognizablesymptom of an existing allergic disease or asthma.

In another embodiment, the diseases or conditions include bacterial orviral infection. An individual having a bacterial or viral infection isan individual with a recognizable symptom of an existing bacterial orviral infection.

In one embodiment, the subject has a viral infection. In a furtherembodiment, the viral infection is a herpes virus infection, a hepatitisvirus infection, a West Nile virus infection, a flavivirus, an influenzainfection, a rhinovirus infection, a papillomavirus infection, aparamyxovirus infection, or a parainfluenza virus infection. In afurther embodiment, the viral infection infects the central nervoussystem of said subject. In still a further embodiment, the viralinfection causes viral encephalitis or viral meningitis.

In one embodiment, the subject has a bacterial infection. A non-limitinglist of bacterial infections treatable with the subject particles of thecurrent invention include staphylococcus infections, streptococcusinfections, mycobacterial infections, bacillus infections, Salmonellainfections, Vibrio infections, spirochete infections, and Neisseriainfections. Preferred are bacteria that infect the central nervoussystem of the subject. Most preferred are bacteria that causeencephalitis or meningitis.

In one embodiment, the method of the invention induces immune tolerancewhen administered to a subject with a bacterial or viral infection. In afurther embodiment, the method ameliorates or dampens an inflammatoryimmune response when administered to a subject with a bacterial or viralinfection.

In yet another embodiment, the subject is a transplant recipient.Transplantation refers to the transfer of a tissue sample or graft froma donor individual to a recipient individual, and is frequentlyperformed on human recipients who need the tissue in order to restore aphysiological function provided by the tissue. Tissues that aretransplanted include (but are not limited to) whole organs such askidney, liver, heart, lung; organ components such as skin grafts and thecornea of the eye; and cell suspensions such as bone marrow cells andcultures of cells selected and expanded from bone marrow or circulatingblood, and whole blood transfusions.

A serious potential complication of any transplantation ensues fromantigenic differences between the host recipient and the engraftedtissue. Depending on the nature and degree of the difference, there maybe a risk of an immunological assault of the graft by the host, or ofthe host by the graft, or both, may occur. The extent of the risk isdetermined by following the response pattern in a population ofsimilarly treated subjects with a similar phenotype, and correlating thevarious possible contributing factors according to well acceptedclinical procedures. The immunological assault may be the result of apreexisting immunological response (such as preformed antibody), or onethat is initiated about the time of transplantation (such as thegeneration of TH cells). Antibody, T helper (TH) cells, or cytotoxic T(Tc) cells may be involved in any combination with each other and withvarious effector molecules and cells. However, the antigens which areinvolved in the immune response are generally not known, thereforeposing difficulties in designing antigen-specific therapies or inducingantigen-specific tolerance. The modified particles of the currentinvention are particularly useful in preventing the rejection of organsbecause no attached peptides or antigens need to be conjugated to themodified particles in order for the particles to be effective ininducing tolerance or ameliorate an inflammatory immune response.

Certain embodiments of the invention relate to decreasing the risk ofhost versus graft disease, leading to rejection of the tissue graft bythe recipient. The treatment may be performed to prevent or reduce theeffect of a hyperacute, acute, or chronic rejection response. Treatmentis preferentially initiated sufficiently far in advance of thetransplant so that tolerance will be in place when the graft isinstalled; but where this is not possible, treatment can be initiatedsimultaneously with or following the transplant. Regardless of the timeof initiation, treatment will generally continue at regular intervalsfor at least the first month following transplant. Follow-up doses maynot be required if a sufficient accommodation of the graft occurs, butcan be resumed if there is any evidence of rejection or inflammation ofthe graft. Of course, the tolerization procedures of this invention maybe combined with other forms of immunosuppression to achieve an evenlower level of risk.

In another embodiment, the diseases or conditions include unwantedimmune activation, such as atherosclerosis, ischemic reperfusion injury,and myocardial infarction.

In yet another embodiment, the invention relates to treatment ofpathological conditions pertaining to an unwanted hypersensitivity. Thehypersensitivity can be any one of types I, II, III, and IV, Immediate(type I) hypersensitivity. The frequency of administration willtypically correspond with the timing of allergen exposure. Suitableanimal models are known in the art (for example, Gundel et al., Am. Rev.Respir. Dis., 146:369, 1992, Wada et al, J. Med. Chem., 39:2055, 1996;and WO 96/35418).

In certain embodiments, treatable diseases or conditions include thoseinitiated by inflammatory monocytes, autoimmunity, cardiovasculardisease (such as cardiac ischemia, or ischemia-reperfusion injuryfollowing cardiac infarction and transplantation), viral encephalitis,multiple sclerosis (MS), inflammatory bowel disease (IBD), peritonitis,lethal flavivirus encephalitis, immunopathological viral infections(including Influenza and West Nile Virus (WNV)), rheumatoid arthritis,HIV encephalitis, chronic liver disease, atherosclerosis, cardiacinfarction, experimental autoimmune encephalomyelitis (EAE) and itscorresponding diseases, Colitis, ulcerative colitis, etc.

In certain embodiments, the microparticle or nanoparticle of theinvention (e.g., those produced with the methods of the invention) canbe used in combination with a second therapeutic that is effective fortreating any one of the treatable conditions.

In certain embodiments, the subject is a human patient. In certainembodiments, the subject is a non-human mammal, such as a non-humanprimate, a livestock animal (horse, mule, cattle, bull, cow, sheep,goat, pig, camel, etc.), a rodent (rabbit, hamster, mouse, rat, etc.),or a pet (cat, dog).

In one embodiment, the method includes administering the subjectcomposition or pharmaceutical composition comprising the subjectmicroparticles or nanoparticles (e.g., the carboxylated particles) byany suitable means or routes, such as orally, nasally, intravenously,intramuscularly, ocularly, transdermally, or subcutaneously. In aparticular embodiment, the particles are administered nasally. In stillanother embodiment, the particles are administered intravenously.

The particles of the present invention can be given in any doseeffective to dampen the inflammatory immune response in a subject inneed thereof or to treat a bacterial or viral infection in a subject inneed thereof. In certain embodiments, about 10² to about 10²⁰ particlesare provided to the individual. In a further embodiment, between about10³ to about 10¹⁵ particles are provided. In yet a further embodiment,between about 10⁶ to about 10¹² particles are provided. In still afurther embodiment, between about 10⁸ to about 10¹⁰ particles areprovided. In one embodiment, the preferred dose is 0.1% solids/ml.Therefore, for 0.5 μm beads, a preferred dose is approximately 4×10⁹beads, for 0.05 μm beads, a preferred dose is approximately 4×10¹²beads, for 3 μm beads, a preferred dose is 2×10⁷ beads. However, anydose that is effective in treating the particular condition to betreated is encompassed by the current invention.

In certain embodiments, the subject composition or subjectpharmaceutical composition containing the subject microparticles ornanoparticles (e.g., carboxylated particles) induces immune tolerancewhen administered to the subject in need thereof.

In a further embodiment, the subject composition or subjectpharmaceutical composition containing the subject microparticles ornanoparticles (e.g., carboxylated particles) ameliorates an inflammatoryimmune response when administered to the subject in need thereof.

Efficacy Tests

The effectiveness of the subject microparticles and nanoparticlesagainst the treatable diseases and conditions can be tested using anumber of efficacy tests, including suitable animal models.

A proxy for tolerogenic activity is the ability of a particle tostimulate the production of an appropriate cytokine at the target site.The immunoregulatory cytokine released by T suppressor cells at thetarget site is thought to be TGF-β (Miller et al., Proc. Natl. Acad.Sci. USA, 89:421, 1992). Other factors that may be produced duringtolerance are the cytokines IL-4 and IL-10, and the mediator PGE. Incontrast, lymphocytes in tissues undergoing active immune destructionsecrete cytokines such as IL-1, IL-2, IL-6, and IFNγ. Hence, theefficacy of a subject particle can be evaluated by measuring its abilityto stimulate the appropriate type of cytokines.

For example, a rapid screening test for a subject particle, effectivemucosal binding components, effective combinations, or effective modesand schedules of mucosal administration can be conducted using animalmodel systems. Animals are treated at a mucosal surface with the testparticle composition, and at some time are challenged withadministration of the disease causing antigen or an infectious agent.Spleen cells are isolated, and cultured in vitro in the presence of thedisease causing antigen or an antigen derived from the infectious agentat a concentration of about 50 μg/mL. Cytokine secretion into the mediumcan be quantified by standard immunoassay.

The ability of the subject particles to suppress the activity of cellscan be determined using cells isolated from an animal immunized with themodified particles, or by creating a cell line responsive to a diseasecausing antigen or viral antigen target antigen (Ben-Nun et al., Eur. J.Immunol., 11195, 1981). In one variation of this experiment, thesuppressor cell population is mildly irradiated (about 1000 to 1250rads) to prevent proliferation, the suppressors are co-cultured with theresponder cells, and then tritiated thymidine incorporation (or MTT) isused to quantitate the proliferative activity of the responders. Inanother variation, the suppressor cell population and the responder cellpopulation are cultured in the upper and lower levels of a dual chambertranswell culture system (Costar, Cambridge Mass.), which permits thepopulations to co-incubate within 1 mm of each other, separated by apolycarbonate membrane (WO 93/16724). In this approach, irradiation ofthe suppressor cell population is unnecessary, since the proliferativeactivity of the responders can be measured separately.

The effectiveness of compositions and modes of administration fortreatment of specific disease can also be elaborated in a correspondinganimal disease model. The ability of the treatment to diminish or delaythe symptomatology of the disease is monitored at the level ofcirculating biochemical and immunological hallmarks of the disease,immunohistology of the affected tissue, and gross clinical features asappropriate for the model being employed. Non-limiting examples ofanimal models that can be used for testing are included below.

For example, animal models for the study of autoimmune disease are knownin the art. Animal models which appear most similar to human autoimmunedisease include animal strains which spontaneously develop a highincidence of the particular disease. Examples of such models include,but are not limited to, the non-obese diabetic (NOD) mouse, whichdevelops a disease similar to type 1 diabetes, and lupus-like diseaseprone animals, such as New Zealand hybrid, MRL-Faslpr and BXSB mice.Animal models in which an autoimmune disease has been induced include,but are not limited to, experimental autoimmune encephalomyelitis (EAE),which is a model for multiple sclerosis, collagen-induced arthritis(CIA), which is a model for rheumatoid arthritis, and experimentalautoimmune uveitis (EAU), which is a model for uveitis. Animal modelsfor autoimmune disease have also been created by genetic manipulationand include, for example, IL-2/IL-10 knockout mice for inflammatorybowel disease, Fas or Fas ligand knockout for SLE, and IL-1 receptorantagonist knockout for rheumatoid arthritis.

The invention contemplates modulation of tolerance by modulating TH1response, TH2 response, TH17 response, or a combination of theseresponses. Modulating TH1 response encompasses changing expression of,e.g., interferon-gamma. Modulating TH2 response encompasses changingexpression of, e.g., any combination of IL-4, IL-5, IL-10, and IL-13.Typically, an increase (decrease) in TH2 response will comprise anincrease (decrease) in expression of at least one of IL-4, IL-5, IL-10,or IL-13; more typically an increase (decrease) in TH2 response willcomprise an increase in expression of at least two of IL-4, IL-5, IL-10,or IL-13, most typically an increase (decrease) in TH2 response willcomprise an increase in at least three of IL-4, IL-5, IL-10, or IL-13,while ideally an increase (decrease) in TH2 response will comprise anincrease (decrease) in expression of all of IL-4, IL-5, IL-10, andIL-13. Modulating TH17 encompasses changing expression of, e.g.,TGF-beta, IL-6, IL-21 and IL-23, and effects levels of IL-17, IL-21 andIL-22.

Tolerance to autoantigens and autoimmune disease is achieved by avariety of mechanisms including negative selection of self-reactive Tcells in the thymus and mechanisms of peripheral tolerance for thoseautoreactive T cells that escape thymic deletion and are found in theperiphery. Examples of mechanisms that provide peripheral T celltolerance include “ignorance” of self antigens, anergy orunresponsiveness to autoantigen, cytokine immune deviation, andactivation-induced cell death of self-reactive T cells. In addition,regulatory T cells have been shown to be involved in mediatingperipheral tolerance. See, for example, Walker et al. (2002) Nat. Rev.Immunol., 2:11-19; Shevach et al. (2001) Immunol. Rev., 182:58-67. Insome situations, peripheral tolerance to an autoantigen is lost (orbroken) and an autoimmune response ensues. For example, in an animalmodel for EAE, activation of antigen presenting cells (APCs) through TLRinnate immune receptors was shown to break self-tolerance and result inthe induction of EAE (Waldner et al. (2004) 1 Clin. Invest.,113:990-997).

Accordingly, in some embodiments, the invention provides methods forincreasing antigen presentation while suppressing or reducing TLR7/8,TLR9, and/or TLR 7/8/9 dependent cell stimulation. As described herein,administration of particular subject particles results in antigenpresentation by DCs or APCs while suppressing the TLR 7/8, TLR9, and/orTLR7/8/9 dependent cell responses associated with immunostimulatorypolynucleotides. Such suppression may include decreased levels of one ormore TLR-associated cytokines.

The subject invention also provides novel compounds that have biologicalproperties useful for the treatment of Mac-1 and LFA-1 mediateddisorders.

Pharmaceutical Composition

One aspect of the present invention provides pharmaceutical compositionswhich comprise the subject microparticles and nanoparticles, andoptionally comprise a pharmaceutically acceptable carrier. In certainembodiments, these compositions optionally further comprise one or moreadditional therapeutic agents. Alternatively, the subject particles ofthe current invention may be administered to a patient in need thereofin combination with the administration of one or more other therapeuticagents. For example, additional therapeutic agents for conjointadministration or inclusion in a pharmaceutical composition with acompound of this invention may be an approved anti-inflammatory agent,or it may be any one of a number of agents undergoing approval in theFood and Drug Administration that ultimately obtain approval for thetreatment of any disorder characterized by an uncontrolled inflammatoryimmune response or a bacterial or viral infection. It will also beappreciated that certain of the subject particles of present inventioncan exist in free form for treatment, or where appropriate, as apharmaceutically acceptable derivative thereof.

In certain embodiments, the pharmaceutical compositions of the presentinvention additionally comprise a pharmaceutically acceptable carrier,which, as used herein, includes any and all solvents, diluents, or otherliquid vehicle, dispersion or suspension aids, surface active agents,isotonic agents, thickening or emulsifying agents, preservatives, solidbinders, lubricants and the like, as suited to the particular dosageform desired. Remington's Pharmaceutical Sciences, Sixteenth Edition, E.W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses variouscarriers used in formulating pharmaceutical compositions and knowntechniques for the preparation thereof. Except insofar as anyconventional carrier medium is incompatible with the compounds of theinvention, such as by producing any undesirable biological effect orotherwise interacting in a deleterious manner with any othercomponent(s) of the pharmaceutical composition, its use is contemplatedto be within the scope of this invention.

Some examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, sugars such aslactose, glucose and sucrose; starches such as corn starch and potatostarch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose and cellulose acetate; powdered tragacanth;malt; gelatin; talc; excipients such as cocoa butter and suppositorywaxes; oils such as peanut oil, cottonseed oil; safflower oil, sesameoil; olive oil; corn oil and soybean oil; glycols; such as propyleneglycol; esters such as ethyl oleate and ethyl laurate; agar; bufferingagents such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogenfree water; isotonic saline; Ringer's solution; ethyl alcohol,and phosphate buffer solutions, as well as other non-toxic compatiblelubricants such as sodium lauryl sulfate and magnesium stearate, as wellas coloring agents, releasing agents, coating agents, sweetening,flavoring and perfuming agents, preservatives and antioxidants can alsobe present in the composition, according to the judgment of theformulator.

Liquid dosage forms for oral administration include, but are not limitedto, pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active compounds,the liquid dosage forms may contain inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor, and sesame oils),glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof. Besides inert diluents,the oral compositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S. P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of a drug, it is often desirable to slowthe absorption of the drug from subcutaneous or intramuscular injection.This may be accomplished by the use of a liquid suspension orcrystalline or amorphous material with poor water solubility.

The rate of absorption of the drug then depends upon its rate ofdissolution that, in turn, may depend upon crystal size and crystallineform. Alternatively, delayed absorption of a parenterally administereddrug form is accomplished by dissolving or suspending the drug in an oilvehicle. Injectable depot forms are made by forming microencapsulematrices of the drug in biodegradable polymers such aspolylactide-polyglycolide. Depending upon the ratio of drug to polymerand the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude (poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissues.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the modifiedparticles are mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid; b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia; c) humectants such as glycerol; d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate; e) solutionretarding agents such as paraffin; f) absorption accelerators such asquaternary ammonium compounds; g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate; h) absorbents such as kaolinand bentonite clay; and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like. The solid dosage forms of tablets, dragees, capsules, pills,and granules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions that can be usedinclude polymeric substances and waxes. Solid compositions of a similartype may also be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polyethylene glycols and the like.

The microparticles and nanoparticles can also be in micro-encapsulatedform with one or more excipients as noted above. The solid dosage formsof tablets, dragees, capsules, pills, and granules can be prepared withcoatings and shells such as enteric coatings, release controllingcoatings and other coatings well known in the pharmaceutical formulatingart. In such solid dosage forms the active compound may be admixed withat least one inert diluent such as sucrose, lactose and starch. Suchdosage forms may also comprise, as in normal practice, additionalsubstances other than inert diluents, e.g., tableting lubricants andother tableting aids such as magnesium stearate and microcrystallinecellulose. In the case of capsules, tablets and pills, the dosage formsmay also comprise buffering agents. They may optionally containopacifying agents and can also be of a composition that they release themodified particles only, or preferentially, in a certain part of theintestinal tract, optionally, in a delayed manner. Examples of embeddingcompositions which can be used include polymeric substances and waxes.

The present invention encompasses pharmaceutically acceptable topicalformulations of the carboxylated microparticles and nanoparticles. Theterm “pharmaceutically acceptable topical formulation,” as used herein,means any formulation which is pharmaceutically acceptable forintradermal administration of the subject microparticles/nanoparticlesby application of the formulation to the epidermis. In certainembodiments of the invention, the topical formulation comprises acarrier system. Pharmaceutically effective carriers include, but are notlimited to, solvents (e.g., alcohols, poly alcohols, water), creams,lotions, ointments, oils, plasters, liposomes, powders, emulsions,microemulsions, and buffered solutions (e.g., hypotonic or bufferedsaline) or any other carrier known in the art for topicallyadministering pharmaceuticals. A more complete listing of art-knowncarriers is provided by reference texts that are standard in the art,for example, Remington's Pharmaceutical Sciences, 16th Edition, 1980 and17th Edition, 1985, both published by Mack Publishing Company, Easton,Pa., the disclosures of which are incorporated herein by reference intheir entireties. In certain other embodiments, the topical formulationsof the invention may comprise excipients. Any pharmaceuticallyacceptable excipient known in the art may be used to prepare theinventive pharmaceutically acceptable topical formulations.

Examples of excipients that can be included in the topical formulationsof the invention include, but are not limited to, preservatives,antioxidants, moisturizers, emollients, buffering agents, solubilizingagents, other penetration agents, skin protectants, surfactants, andpropellants, and/or additional therapeutic agents used in combination tothe modified particles. Suitable preservatives include, but are notlimited to, alcohols, quaternary amines, organic acids, parabens, andphenols. Suitable antioxidants include, but are not limited to, ascorbicacid and its esters, sodium bisulfite, butylated hydroxytoluene,butylated hydroxyanisole, tocopherols, and chelating agents like EDTAand citric acid. Suitable moisturizers include, but are not limited to,glycerine, sorbitol, polyethylene glycols, urea, and propylene glycol.Suitable buffering agents for use with the invention include, but arenot limited to, citric, hydrochloric, and lactic acid buffers. Suitablesolubilizing agents include, but are not limited to, quaternary ammoniumchlorides, cyclodextrins, benzyl benzoate, lecithin, and polysorbates.Suitable skin protectants that can be used in the topical formulationsof the invention include, but are not limited to, vitamin E oil,allatoin, dimethicone, glycerin, petrolatum, and zinc oxide.

In certain embodiments, the pharmaceutically acceptable topicalformulations of the invention comprise at least the carboxylatedmicroparticles and nanoparticles and a penetration enhancing agent. Thechoice of topical formulation will depend on several factors, includingthe condition to be treated, the physicochemical characteristics of theparticles and other excipients present, their stability in theformulation, available manufacturing equipment, and costs constraints.As used herein the term “penetration enhancing agent” means an agentcapable of transporting a pharmacologically active compound through thestratum corneum and into the epidermis or dermis, preferably, withlittle or no systemic absorption. A wide variety of compounds have beenevaluated as to their effectiveness in enhancing the rate of penetrationof drugs through the skin. See, for example, Percutaneous PenetrationEnhancers, Maibach H. I. and Smith H. E. (eds.), CRC Press, Inc., BocaRaton, Fla. (1995), which surveys the use and testing of various skinpenetration enhancers, and Buyuktimkin et al., Chemical Means ofTransdermal Drug Permeation Enhancement in Transdermal and Topical DrugDelivery Systems, Gosh T. K., Pfister W. R., Yum S. I. (Eds.),Interpharm Press Inc., Buffalo Grove, 111 (1997). In certain exemplaryembodiments, penetration agents for use with the invention include, butare not limited to, triglycerides (e.g., soybean oil), aloe compositions(e.g., aloe vera gel), ethyl alcohol, isopropyl alcohol,octolyphenylpolyethylene glycol, oleic acid, polyethylene glycol 400,propylene glycol, N-decylmethylsulfoxide, fatty acid esters (e.g.,isopropyl myristate, methyl laurate, glycerol monooleate, and propyleneglycol monooleate) and N-methylpyrrolidone.

In certain embodiments, the compositions may be in the form ofointments, pastes, creams, lotions, gels, powders, solutions, sprays,inhalants or patches. In certain exemplary embodiments, formulations ofthe compositions according to the invention are creams, which mayfurther contain saturated or unsaturated fatty acids such as stearicacid, palmitic acid, oleic acid, palmito-oleic acid, cetyl or oleylalcohols, stearic acid being particularly preferred. Creams of theinvention may also contain a non-ionic surfactant, for example, polyoxystearate. In certain embodiments, the active component is admixed understerile conditions with a pharmaceutically acceptable carrier and anyneeded preservatives or buffers as may be required. Ophthalmicformulation, eardrops, and eye drops are also contemplated as beingwithin the scope of this invention. Additionally, the present inventioncontemplates the use of transdermal patches, which have the addedadvantage of providing controlled delivery of a compound to the body.Such dosage forms are made by dissolving or dispensing the compound inthe proper medium. As discussed above, penetration enhancing agents canalso be used to increase the flux of the compound across the skin. Therate can be controlled by either providing a rate controlling membraneor by dispersing the compound in a polymer matrix or gel.

The carboxylated microparticles and nanoparticles can be administered byaerosol. This is accomplished by preparing an aqueous aerosol, liposomalpreparation or solid particles containing the modified particles. A nonaqueous (e.g., fluorocarbon propellant) suspension could be used.

Ordinarily, an aqueous aerosol is made by formulating an aqueoussolution or suspension of the agent together with conventionalpharmaceutically acceptable carriers and stabilizers. The carriers andstabilizers vary with the requirements of the particular compound, buttypically include nonionic surfactants (Tweens, Pluronics, orpolyethylene glycol), innocuous proteins like serum albumin, sorbitanesters, oleic acid, lecithin, amino acids such as glycine, buffers,salts, sugars or sugar alcohols. Aerosols generally are prepared fromisotonic solutions.

It will also be appreciated that the carboxylated nanoparticles andmicroparticles and pharmaceutical compositions of the present inventioncan be formulated and employed in combination therapies, that is, thecompounds and pharmaceutical compositions can be formulated with oradministered concurrently with, prior to, or subsequent to, one or moreother desired therapeutics or medical procedures. The particularcombination of therapies (therapeutics or procedures) to employ in acombination regimen will take into account compatibility of the desiredtherapeutics and/or procedures and the desired therapeutic effect to beachieved. It will also be appreciated that the therapies employed mayachieve a desired effect for the same disorder (for example, aninventive compound may be administered concurrently with anotheranti-inflammatory agent), or they may achieve different effects (e.g.,control of any adverse effects).

In certain embodiments, the pharmaceutical compositions containing thecarboxylated particles of the present invention further comprise one ormore additional therapeutically active ingredients (e.g.,anti-inflammatory and/or palliative). For purposes of the invention, theterm “Palliative” refers to treatment that is focused on the relief ofsymptoms of a disease and/or side effects of a therapeutic regimen, butis not curative. For example, palliative treatment encompassespainkillers, anti nausea medications and anti-sickness drugs.

The following examples are given to illustrate the present invention. Itshould be understood, however, that the invention is not to be limitedto the specific conditions or details described in these examples.Throughout the specification, any and all references to a publiclyavailable document, including any U.S. patent or patent applicationpublication, are specifically incorporated by reference.

Examples Example 1. Preparation of Carboxylated PLGA Nanoparticles ViaEmulsification Process with Hyaluronic Acid

0.75 g PLGA (Lakeshore DLG 5050 4.5A) was dissolved in 15 ml methylenechloride. The PLGA solution was mixed with 150 ml 1% polyvinyl alcoholsolution containing 0.2 gram of hyaluronic acid, and homogenized at12,500 rpm for 90 seconds on a NISSEN Homogenizer. The resultingemulsion was poured to a glass container and stirred magnetically at 400rpm for 4 hours to allow the evaporation of the solvent. Thenanoparticles were washed three times with distilled water before theywere lyophilized.

Particle size and zeta potential were determined with a Malvern particlesize analyzer (Worcestershire, UK). The average particle size was foundto be 122.5 nm and zeta potential was −31.2 mV.

Example 2. Preparation of Carboxylated PLGA Nanoparticles ViaEmulsification Process with a Short-Chain PLGA Polymer

0.19 g PLGA (Lactel B6013, inherent viscosity 0.15-0.25 dL/g) wasdissolved in 10 ml methylene chloride. The PLGA solution was mixed with100 ml 1% polyvinyl alcohol solution and homogenized at 18,400 rpm for45 seconds with an IKA T25_digital_ULTRA-TURRAX Homogenizer. Theresulting emulsion was poured to a glass container and stirredmagnetically at 400 rpm for 4 hours to allow the evaporation of thesolvent. The nanoparticles were washed three times with distilled waterbefore they were lyophilized.

Particle size and zeta potential were determined with a Beckman-CoulterLS320 Laser Diffraction Particle Size Analyzer. The average particlesize was found to be 680 nm.

Example 3. Preparation of Carboxylated PLGA Nanoparticles ViaPrecipitation Process with a Short-Chain PLGA Polymer

0.42 g PLGA polymer (Lakeshore DLG 5050 1A, inherent viscosity 0.05-0.15dL/g) was dissolved in 10 ml acetone. This PLGA/acetone solution wasadded using a syringe pump at an addition rate of about 25 mL/hour to 60mL 1 mM NaOH solution. The resulting nanoparticle suspension was mixedwith 1 liter of distilled water and concentrated to approximately 20 mLwith a tangential flow filtration device and a 500 kDa molecular weightcut-off module. The concentrated nanoparticle suspension waslyophilized.

Particle size and zeta potential were determined with a Malvern particlesize analyzer (Worcestershire, UK). The average particle size was foundto be 230.4 nm and zeta potential was −31.1 mV.

Example 4. Preparation of Carboxylated PLGA Nanoparticles ViaEmulsification Process with a Short-Chain PLGA Polymer Containing TwoTerminal Carboxyl Groups

0.22 g PLGA (Lactel B6013, initiated by glycolic acid) containingterminal COOH groups on both chain ends was dissolved in 10 ml methylenechloride. The PLGA solution was mixed with 150 ml 1% polyvinyl alcoholin 1 mM NaOH solution and homogenized at 18,000 rpm for 60 seconds on aNISSEN Homogenizer. The resulting emulsion was poured to a glasscontainer and stirred magnetically at 500 rpm for 4 hours to allow theevaporation of the solvent. The nanoparticles were washed three timeswith distilled water before lyophilization.

Example 5. Preparation of Carboxylated PLGA Nanoparticles ViaEmulsification Process with Hyaluronic Acid

0.75 g PLGA (Lakeshore DLG 5050 4.5A) is dissolved in 15 ml ethylacetate to form a PLGA solution. The PLGA solution is then mixed with amixture consisting essentially of 138.75 ml 1% polyvinyl alcoholsolution (in water), 11.25 ml of ethyl acetate, and 0.2 gram ofhyaluronic acid, and is homogenized at 12,500 rpm for 90 seconds on aNISSEN Homogenizer. The resulting emulsion is poured to a glasscontainer and stirred magnetically at 400 rpm for 4 hours to allow theevaporation of the solvent. The nanoparticles are washed three timeswith distilled water before they are lyophilized.

1. A method for preparing PLGA microparticles or nanoparticles havingnegative surface charges, said method comprising: (1) dissolving anactive pharmaceutical ingredient (API) in an inner aqueous solvent toform an API solution; (2) dissolving a pharmaceutically acceptable PLGApolymer having an average molecular weight of from about 1,000 to about1,000,000 Da, in an organic solvent to form a polymer solution, whereinthe organic solvent is not miscible or is partially miscible in theinner aqueous solvent; (3) emulsifying the API solution in the polymersolution; (4) dissolving a pharmaceutically acceptable polyacrylic acidand a surfactant; (5) emulsifying the solution of step (3) in the outeraqueous solution to form an emulsion; and (6) removing the organicsolvent to form said PLGA microparticles or nanoparticles havingnegative surface charges; wherein the microparticles or nanoparticleshave a zeta potential of from about −15 mV to −100 mV and wherein thepharmaceutically acceptable polyacrylic acid is incorporated onto and atleast partially into the PLGA microparticles.
 2. The method of claim 1,wherein the surfactant is polyvinyl alcohol.
 3. The method of claim 1,wherein the organic solvent is selected from the group consisting ofmethylene chloride, ethyl acetate, or chloroform, and mixtures thereof.4. The method of claim 1, wherein each emulsifying step compriseshomogenization.
 5. The method of claim 1, wherein the organic solvent isremoved through evaporation.
 6. The method of claim 1, wherein the pH ofthe outer aqueous solution is between about 6 to about
 10. 7. The methodof claim 1, wherein the microparticles or nanoparticles have averageparticle sizes of from about 10 nm to about 100 μm.
 8. The method ofclaim 1, wherein the microparticles or nanoparticles have a zetapotential of from −35 mV to −85 mV.